Human HAC3

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of hHAC3, antibodies to hHAC3, methods of detecting hHAC3, and methods of screening for modulators hyperpolarization-activated cation channels using biologically active hHAC3. The invention further provides, in a computer system, a method of screening for mutations of human HAC3 genes as well as a method for identifying a three-dimensional structure of human HAC3 polypeptides.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 60/129,456, filedApr. 15, 1999, herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofhHAC3, antibodies to hHAC3, methods of detecting hHAC3, and methods ofscreening for modulators hyperpolarization-activated cation channelsusing biologically active hHAC3. The invention further provides, in acomputer system, a method of screening for mutations of human HAC3 genesas well as a method for identifying a three-dimensional structure ofhuman HAC3 polypeptides.

BACKGROUND OF THE INVENTION

A. General Background to Cation Channels

Cation channels are a diverse group of proteins that regulate the flowof cations across cellular membranes. The selectivity of a cationchannel for particular cations typically varies with the valency of thecations, as well as the specificity of a given channel for a particularcation. Some cation channels display almost no selectivity for cationswith the same valence (see, e.g., Saitow et al, Biochim Biophys Acta1327(1):52-60 (1997)). Other channels are clearly selective forparticular cations but are permeable to other cations to varying degrees(see, e.g., Park & MacKinnon, Biochemistry 34(41):13328-33 (1995) andGauss et al., Nature 393(6685):583-7 (1998)).

Cation channels are involved in a number of physiological processes,including regulation of heartbeat, dilation of arteries, release ofinsulin, excitability of nerve cells, transduction of sensory stimuli,and regulation of renal electrolyte transport. Cation channels are thusfound in a wide variety of animal cells such as nervous, muscular,glandular, immune, reproductive, sensory, and epithelial tissue. Thesechannels allow the flow of various cations in and/or out of the cellunder certain conditions. For example, the inward flow of cations uponopening of these channels makes the interior of the cell more positive,thus depolarizing the cell. These channels are regulated, e.g., bycalcium sensitivity, voltage-gating, cyclic nucleotides or othersecondary messengers, extracellular ligands, and ATP-sensitivity.

Certain classes of cation channels are formed by four alpha subunits andcan be homomeric (made of identical alpha subunits) or heteromeric (madeof two or more distinct types of alpha subunits). Some cation channelsmay contain other structurally distinct auxiliary, or beta, subunits.These subunits do not form potassium channels themselves, but insteadmodify the functional properties of channels formed by the alphasubunits. For example, the Kv beta subunits are cytoplasmic and areknown to increase the surface expression of Kv channels and/or modifytheir inactivation kinetics (Heinemann et al., J Phsyiol. (Lond);493:625-633; 1996 and Shi et al., Neuron 16(4):843-852, 1996). Inanother example, the KQT family beta subunit, minK, primarily changesactivation kinetics (Sanguinetti et al., Nature 384:80-83, 1996).

B. Hyperpolarization-activated Cation Channels: HAC1 and HAC2.

Specialized cells in the heart and brain can create rhythmic activitydue in a large part to a depolarizing mixed sodium/potassium currentknown as I_(h) (see, e.g., Santoro et al., Cell 93:717-729 (1998)). Thispacemaker current is generated by hypolarization activated channels thatare present in the heart (see, e.g., DiFrancesco, Ann. Rev Physiol.55:455-72 (1993) and brain (see, e.g., Papa, Ann. Rev. Physiol.58:299-327 (1996). In addition to contributing directly to rhythmicactivity in the brain and heart, these channels may contributesignificantly to resting membrane potentials in neurons and other celltypes from a variety of tissues.

Recently a family of hyperpolarization-activated channels, given theacronym HAC, was isolated from mouse (see, Ludwig et al., Nature393:587-91 (1998)). Ludwig et al. reported isolating three different ionchannels (mHAC1, mHAC2 and mHAC3). The mouse HAC proteins are members ofthe voltage-gated cation channel super family and also have a cyclicnucleotide binding domain capable of binding cAMP and cGMP. Mouse HAC1exhibits the general properties of I_(h) and may be responsible forpacemaker activity.

Another group also identified the same gene family, in this instanceidentified by the acronym BCNG. For instance, the BCNG-1 (HAC2) ionchannel was isolated from mouse cells and is expressed in the brain(see, e.g., Santoro et al., Proc. Natl. Sci. USA 94:14815-20 (1997)).The human BCNG-2/HAC1 and BCNG-1/HAC2 have also been cloned (see, e.g.,Santoro et al., Cell 93:717-729 (1998)). Since then, several relatedmouse genes (e.g., BCNG-1/HAC2, partial BCNG2/HAC1, partial BCNG3/HAC4,and partial BCNG4/HAC3) with expression in various tissues, includingheart and brain, have been isolated (see, e.g., Santoro et al., Cell93:717-729 (1998)).

Phylogenetic analysis indicates that mHAC3 is more distantly related tomHAC 1 or mHAC2 than are mHAC 1 and mHAC2 to each other. Human HAC3 hasnot been previously isolated. Isolation of human HAC3 is thereforedesirable, to better understand the physiology of HAC3 in humans and forthe development of therapeutic and diagnostic applications to diseasesrelated to hHAC3 in humans.

SUMMARY OF THE INVENTION

The current invention provides the first isolation and characterizationof the human HAC3 cation channel, which has neither been previouslycloned nor identified. The present invention provides both thenucleotide and amino acid sequence of hHAC3, as well as methods ofassaying for modulators of hHAC3, antibodies to hHAC3, and methods ofdetecting hHAC3 nucleic acids and proteins.

The present invention provides an isolated nucleic acid encoding apolypeptide monomer comprising an alpha subunit of a cation channelwherein the polypeptide monomer has two attributes. First, thepolypeptide monomer forms, with at least one additional HAC alphasubunit, a cation channel having the characteristic of activation uponhyperpolarization. Second, the polypeptide monomer has an amino acidsequence that has greater than about 75% identity to an N-terminalregion (amino acids 1-50) of a human HAC3 amino acid sequence (e.g., SEQID NO:1) or greater than about 90% identity to amino acids 640-775 of ahuman HAC3 amino acid sequence (e.g., SEQ ID NO:1).

In one embodiment of the invention, the nucleic acid encodes SEQ IDNO:1. In another embodiment, the nucleic acid has a nucleotide sequenceof SEQ ID NO:2. In yet another embodiment, the nucleic acid is a splicevariant of SEQ ID NO:2.

In one embodiment of the invention includes a nucleic acid that isamplified by primers that selectively hybridize under stringentconditions to the same sequence as any two primers selected fromCAGCCATGGAGGCAGAGCAGCGGC (SEQ ID NO:3), GGAGGAGATCTTTCACATGACATACGAC(SEQ ID NO:4), AGTAGGATCCATCGGTGAGGCGTG (SEQ ID NO:5), andTTACATGTTGGCAGAAAGCTGGAGACC (SEQ ID NO:6).

In one embodiment of the invention, the nucleic acid selectivelyhybridizes under moderately stringent hybridization conditions to thenucleotide of SEQ ID NO:2.

In one embodiment of the invention, the nucleic acid has a nucleotidesequence that has greater than about 90% identity to SEQ ID NO:2. Inanother embodiment, the nucleic acid encodes a polypeptide having anamino acid sequence that has greater than about 96% identity to SEQ IDNO:1.

The present invention also provides an isolated protein monomercomprising an alpha subunit of a cation channel wherein the polypeptidemonomer 1) forms, with at least one additional HAC alpha subunit, acation channel having the characteristic of activation uponhyperpolarization, and 2) has an amino acid sequence that has greaterthan about 75% identity to an N-terminal region (amino acids 1-50) orgreater than about 90% identity to amino acids 640-775 of a human HAC3amino acid sequence.

In one embodiment of the invention, the polypeptide monomer specificallybinds to antibodies generated against SEQ ID NO:1.

In one embodiment, the isolated peptide monomer has an amino acidsequence of SEQ ID NO:1. In different embodiments, the isolated peptidemonomer comprises either an alpha subunit of a homomeric or aheteromeric cation channel. In another embodiment, the isolatedpolypeptide monomer has a molecular weight between about 84 kDa andabout 95 kDa. In yet another embodiment, the isolated polypeptidemonomer has greater than about 96% identity to SEQ ID NO:1.

One aspect of the invention includes an antibody that selectively bindsto a polypeptide monomer that 1) forms, with at least one additional HACalpha subunit, a cation channel having the characteristic of activationupon hyperpolarization, and 2) has an amino acid sequence that hasgreater than about 75% identity to an N-terminal region (amino acids1-50) or greater than about 90% identity to amino acids 640-775 of ahuman HAC3 amino acid sequence (e.g., SEQ ID NO:1).

The invention also provides for an expression vector comprising anucleic acid encoding a polypeptide monomer comprising a subunit of acation channel, wherein the cation channel (i) has the characteristic ofactivation upon hyperpolarization; and (ii) comprises a polypeptidemonomer having an amino acid sequence that has greater than about 96%amino acid sequence identity to a human HAC3 amino acid sequence. In oneembodiment, a host cell is transfected with the expression vector.

The invention also provides a method for identifying a compound thatincreases or decreases ion flux through a hyperpolarization-activatedcation channel. The method comprises two steps. The first step comprisescontacting the compound with a HAC polypeptide monomer. The polypeptidemonomer 1) forms, with at least one additional HAC alpha subunit, acation channel having the characteristic of activation uponhyperpolarization, and 2) has an amino acid sequence that has greaterthan about 75% identity to an N-terminal region (amino acids 1-50) orgreater than about 90% identity to amino acids 640-775 of a human HAC3amino acid sequence (e.g., SEQ ID NO:1). The second step of the methodcomprises determining the functional effect of the compound upon thecation channel.

In one embodiment, the functional effect is a physical effect or afunctional effect. In another embodiment, the polypeptide is expressedin a eukaryotic host cell or cell membrane.

In one embodiment of the method, the functional effect is determined bymeasuring ion flux, or changes in current, voltage, ion concentrations,or yeast viability. In another embodiment, the isolated polypeptidemonomer is recombinant. The method provides for a polypeptide monomercomprising an alpha subunit of either a homomeric or a heteromericcation channel. Finally, in one aspect of the method, the polypeptidemonomer has the amino acid sequence of SEQ ID NO:1.

In another aspect, the present invention provides a method of modulatingion flux through a human HAC channel, the method comprising the step ofcontacting the human HAC channel with a therapeutically effective amountof a compound identified as described above

In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through aHAC potassium channel comprising a human HAC polypeptide, the methodcomprising the steps of: (i) entering into a computer system an aminoacid sequence of at least 50 acids of a human HAC polypeptide or atleast 150 nucleotides of a nucleic acid encoding the human HACpolypeptide, the human HAC polypeptide having an amino acid sequencethat has greater than about 75% identity to amino acids 1-50 of SEQ IDNO:1 or greater than about 90% identity to amino acids 640-775 of SEQ IDNO:1; (ii) generating a three-dimensional structure of the polypeptideencoded by the amino acid sequence; (iii) generating a three-dimensionalstructure of the potassium channel comprising the human HAC polypeptide;(iv) generating a three-dimensional structure of the compound; and (v)comparing the three-dimensional structures of the polypeptide and thecompound to determine whether or not the compound binds to thepolypeptide.

The invention also provides for a method of detecting the presence ofHAC3 in a sample. The method comprises the steps of: (i) isolating abiological sample; (ii) contacting the biological sample with a humanHAC3-specific reagent that selectively associates with human HAC3; and,(iii) detecting the level of human HAC3-specific reagent thatselectively associates with the sample.

In one embodiment, the human HAC3-specific reagent is selected from thegroup consisting of: human HAC3 specific antibodies, human HAC3 specificoligonucleotide primers, and human HAC3 nucleic acid probes.

The invention further provides for a method of screening for mutationsof human HAC3 genes using a computer system. The method comprises (i)entering into a computer system a first nucleic acid sequence encodingan hyperpolarization-activated cation channel polypeptide monomer havinga nucleotide sequence of SEQ ID NO:2, and conservatively modifiedversions thereof; (ii) comparing the first nucleic acid sequence with asecond nucleic acid sequence having substantial identity to the firstnucleic acid sequence; and (iii) identifying nucleotide differencesbetween the first and second nucleic acid sequences. In one embodimentof this method, the second nucleic acid sequence is associated with adisease state. The invention further provides a computer readablesubstrate comprising the first nucleic sequence as described in theabove-described method. In one embodiment of this composition, thecomputer readable substrate further comprises the second nucleic acid asdescribed in the above-described method.

Finally, the invention also provides a method for identifying athree-dimensional structure of human HAC3 polypeptide monomers. Themethod comprises (i) entering into a computer system an amino acidsequence of at least 60 amino acids of a polypeptide monomer or at least180 nucleotides of a gene encoding the polypeptide monomer, thepolypeptide monomer having an amino acid sequence of SEQ ID NO:1, andconservatively modified versions thereof; and (ii) generating athree-dimensional structure of the polypeptide monomer encoded by theamino acid sequence.

In one embodiment of this method, the amino acid sequence is a primarystructure and the generating step includes the steps of (i) forming asecondary structure from said primary structure using energy termsdetermined by the primary structure; and (ii) forming a tertiarystructure from said secondary structure using energy terms determined bysaid secondary structure. In another embodiment, the generating stepalso includes the step of forming a quaternary structure from thetertiary structure using anisotropic terms encoded by the tertiarystructure. In another aspect of the method, the method also includes thestep of identifying regions of the three-dimensional structure of ahuman HAC3 cation channel protein that bind to ligands and using theregions to identify ligands that bind to the cation channel. A furtheraspect of the invention includes a computer readable substratecomprising the tree dimensional structure derived from theabove-described method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid alignment of human Hac3 with human Hac1 and humanHac2. Identical residues are shaded. The numbers at the left edgeindicated amino acid position. Note that the human Hac2 is missing theamino terminus.

FIG. 2. Northern blot analysis of human Hac3. (A) Traditional northernblot of Hac3. A transcript of approximately 4 Kb is abundant in brainand also present in heart. Larger transcripts (approximately 5 Kb) areseen in brain, heart, liver and kidney. (B) mRNA dot blot northern ofHac3. Expression is most abundant in brain, but is widespread inperipheral tissues. Note that mRNA dot blots are several times moresensitive for detection of message than traditional northerns. Also notethe wide discrepancy between the high level of message detected on thedot blot for small intestine and colon versus the lack of expression onthe traditional northern. These tissues often label more highly on dotblots, and it is possible that this is an effect of poor mRNA qualityfor these tissues.

FIG. 3. Hac3 currents expressed in Xenopus oocytes. (A) Hac3 currentswere elicited with 3.2 second pulses ranging from −70 to −160 mV in 10mV increments from a holding potential of −30 mV. Outward tail currentswere measured at 0 mV. (B) Hac3 currents were elicited by steps to −100mV and tail currents were measured at repolarization steps ranging from−50 to 0 mV in 10 mV increments. The current reverses between −40 and−30 mV suggesting that Hac3 passes both sodium and potassium.

FIG. 4. Hac3 currents are blocked by Cesium. Hac3 currents were elicitedby steps from −30 mV to −140 mV. Tail currents were measured at 0 mV. 2mM Cs⁺ completely blocked the inward Hac3 current measured at −140 mV,but had less effect on the outward tail current. This suggests that theCs⁺ blocking site is in the external mouth of the Hac3 pore.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides for the first time a nucleic acidencoding a human HAC3 alpha subunit, identified and cloned from humantissue. This polypeptide monomer is a member of the HAC family ofpotassium channel monomers and is most closely related to the CNGchannel α-subunits and the “eag” (ether à go-go) subfamily of potassiumchannel monomers. Members of this family are voltage-gated cationchannels with six membrane-spanning segments (S1-S6). These segmentsinclude a voltage sensing S4 segment and an ion conducting pore betweenS5 and S6. Voltage-gated cation channels have significant roles inmaintaining the resting potential and in controlling excitability of acell.

The invention also provides methods of screening for modulators ofhyperpolarization-activated cation channels comprising a hHAC3 alphasubunit. For example, such modulators may alter the voltage-gatingcharacteristic of hHAC. Hyperpolarization activated channels, such asthose comprising hHAC3, have a greater probability of opening when themembrane comprising the channels is hyperpolarized. Modulators ofhyperpolarization-activated channel activity may be useful for treatingvarious pacemaker dysfunctions such as familial sinus rhythm diseases,sick sinus syndrome associated with atrial fibrillation, sinustachycardias and bradycardias as well as ventricular arrhythmias. Themodulators are also useful for treating other disorders involvingabnormal ion flux, e.g., memory and learning disorders, sleepingdisorders, bipolar disease, schizophrenia, CNS disorders such asmigraines, hearing and vision problems, seizures, and as neuroprotectiveagents (e.g., to prevent stroke).

Furthermore, the invention provides assays for hHAC3 activity wherehHAC3 acts as a direct or indirect reporter molecule. Human HAC3 canhave broad application as a reporter molecule in assay and detectionsystems. For instance, hHAC3 can be used as a reporter molecule tomeasure changes in potassium or sodium concentration, membranepotential, current flow, ion flux, transcription, signal transduction,receptor-ligand interactions, second messenger concentrations, in vitro,in vivo, and ex vivo. In one embodiment, hHAC3 can be used as anindicator of current flow in a particular direction (e.g., outward orinward cation flow), and in another embodiment, hHAC3 can be used as anindirect reporter via attachment to a second reporter molecule such asgreen fluorescent protein.

The invention provides for methods of detecting hHAC3 nucleic acid andprotein expression, allowing investigation of the channel diversityprovided by hHAC3, as well as diagnosis of disease caused by pacemakeractivity dysfunction such as familial sinus rhythm diseases, sick sinussyndrome associated with atrial fibrillation, sinus tachycardias,bradycardias and ventricular arrhythmias as well as abnormal ion flux,e.g., memory and learning disorders, sleeping disorders, bipolardisease, schizophrenia, CNS disorders such as migraines, hearing andvision problems, seizures.

Finally, the invention provides for a method of screening for mutationsof hHAC3 genes or proteins. The invention includes, but is not limitedto, methods of screening for mutations in hHAC3 with the use of acomputer. Similarly, the invention provides for methods of identifyingthe three-dimensional structure of hHAC3, as well as the resultingcomputer readable images or data that comprise the three dimensionalstructure of hHAC3. Other methods for screening for mutations of hHACgenes or proteins include high density oligonucleotide arrays, PCR,immunoassays and the like.

Functionally, hHAC3 is an alpha subunit of a voltage-gated cationchannel that is activated upon hyperpolarization. Voltage-gated channelsare heteromeric or homomeric and typically contain four alpha subunitsor monomers, each with six transmembrane domains. Heteromeric channelscomprise one or more hHAC3 alpha subunits along with additional alphasubunits from the HAC family (e.g., HAC1 or HAC2). In addition, suchchannels may comprise one or more auxiliary beta subunits. At itscarboxy terminus, hHAC3 also contains a sequence with similarity tocyclic nucleotide binding proteins. Therefore, it is likely that hHAC3channel activity can be modulated by cyclic nucleotides such as cAMP orcGMP. The presence of hHAC3 in a cation channel may also modulate theactivity of the channel and thus enhance channel diversity. Channeldiversity is also enhanced with alternatively spliced forms of hHAC3.The cation channels may also include an auxiliary beta subunit thatmodulates channel activity and thus enhances channel diversity.

Structurally, the nucleotide sequence of human HAC3 (SEQ ID NO:2)encodes a polypeptide monomer of approximately 775 amino acids with apredicted molecular weight of 85-94 kDa. Human HAC3 contains sixmembrane spanning domains (S1-6), including a voltage sensing domain(S4) and an ion-conduction pore between S5 and S6, as well as a putativecyclic nucleotide binding domain region that has a conserved amino acidsequence. This entire region is located at approximately amino acids 53to 554. Furthermore, hHAC3 contains an N-terminal domain located atamino acids 1 to 50, which provides a means for identifying alleles andconservatively modified variants of hHAC3. Alternatively, hHAC3 can beidentified as a cation channel subunit polypeptide having 90% or moreidentity to the region defined by amino acids 640-775 of SEQ ID NO:1.

The present invention also provides polymorphic variants of hHAC3. Forinstance, in variant #1, an aspartate is substituted for a leucine atposition 545. In variant #2, a valine is substituted for an isoleucineat amino acid position number 37. In variant #3, a threonine issubstituted for an alanine at amino acid position 686. In variant #4, analanine is substituted for a glycine at amino acid position 702.

Specific regions of hHAC3 amino acid and nucleotide sequences may beused to identify conservatively modified or polymorphic variants andalleles of hHAC3. This identification can be made in vitro, e.g., understringent hybridization conditions and sequencing, or by using thesequence information in a computer system for comparison with othernucleotide sequences. Amino acid identity of approximately at least 96%or above, preferably 98%, most preferably 99% or above to the entirehHAC3 polypeptide (SEQ ID NO:1) or a portion thereof, typicallydemonstrates that a protein is a polymorphic variant or allele of hHAC3.The first 50 amino acid residues of SEQ ID NO:1 displays considerablevariance relative to the mouse HAC3 N-terminus. Since this region issignificantly different from other known HAC sequences, the first 50amino acids of SEQ ID NO:1 are preferably used to differentiatesequences related to human HAC3 from HAC sequences from other species.Therefore, an amino acid identity of approximately 75% or above,preferably 80%, and most preferably 90% or above to the first 50 aminoacids of SEQ ID NO:1 peptide demonstrates that a protein is aconservatively modified or polymorphic variant or allele of hHAC3.Alternatively, the conserved region of amino acids 640-775 of SEQ IDNO:1 can be used to identify conservatively modified variants, alleles,and polymorphic variants of hHAC3. Amino acid sequence identity of 90%or more to this conserved region demonstrates that a protein is aconservatively modified or polymorphic variant or allele of hHAC3.

Nucleotide identity of approximately at least 90%, preferably 95% andmost preferably 98% or above to the entire hHAC3 nucleic acid sequence(SEQ ID NO:2) or portions thereof, typically demonstrates that a nucleicacid is a conservatively modified or polymorphic variant or allele ofhHAC3. Alternatively, hHAC3 can be identified as a cation channelsubunit polypeptide having 90% or more identity to the region defined byamino acids 640-775 of SEQ ID NO:1. Sequence comparison is performedusing the sequence comparison algorithms discussed below, using thedefault parameters described below. Antibodies that bind specifically tothe HAC3 subunit can also be used to identify alleles, conservativelymodified or polymorphic variants. Finally, analysis of the threedimensional structure of the hHAC3 polypeptide can be used to predictalleles of hHAC3 that have conserved function.

Conservatively modified or polymorphic variants and alleles of hHAC3 areconfirmed by expressing the putative hHAC3 polypeptide monomer eitheralone or co-expressed with another cation channel subunit and examiningwhether the monomer forms a cation channel. Functional assays may beused to determine the characteristics of the cation channels formed insuch ways. One assay is to determine changes in cellular polarization bymeasuring changes in current (thereby measuring changes in polarization)with voltage-clamp and patch-clamp techniques, e.g., “the cell-attached”mode, “the inside-out” mode, and “the whole cell” mode (see, e.g.,Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). This assay isused to demonstrate that a cation channel comprising a polypeptidemonomer having about 96% or greater, preferably 98% or greater aminoacid identity to the entire sequence of hHAC3 or a portion thereof is aspecies of hHAC3 because the subunit shares the same functionalcharacteristics. Typically, hHAC3 monomers having the amino acidsequence of SEQ ID NO:1 are used as positive controls in comparison tothe putative hHAC3 protein to demonstrate the identification of apolymorphic variant or allele of hHAC3.

Human HAC3 nucleotide and amino acid sequence information may also beused to construct models of a hyperpolarization-activated cation channelin a computer system. These models are subsequently used to identifycompounds that can activate or inhibit a hyperpolarization-activatedcation channel comprising hHAC3. Such compounds that modulate theactivity of channels comprising hHAC3 can be used to investigate therole of hHAC3 in modulation of channel activity and in channeldiversity.

The identification and cloning of hHAC3 for the first time provides ameans for assaying for inhibitors and activators of humanhyperpolarization-activated cation channels such as cation channelscomprising hHAC3. Biologically active hHAC3 is useful for testinginhibitors and activators of cation channels comprising hHAC3 and otherhyperpolarization-activated cation channels using in vivo and in vitroexpression that measure, e.g., changes in voltage or current. Suchactivators and inhibitors, identified using a voltage-gated cationchannel comprising at least one hHAC3 monomer, can be used to furtherstudy, e.g., regulation of cation channels activated uponhyperpolarization, channel kinetics, and conductance properties of suchchannels. These activators and inhibitors are also useful aspharmaceutical agents for treating diseases involving pacemakerdysfunctions such as familial sinus rhythm diseases, sick sinus syndromeassociated with atrial fibrillation, sinus tachycardias, bradycardiasand ventricular arrhythmias as well as abnormal ion flux, e.g., memoryand learning disorders, sleeping disorders, bipolar disease,schizophrenia, CNS disorders, as described above.

Methods of detecting hHAC3 and expression of channels comprising thehHAC3 monomers are also useful for diagnostic applications for diseasesinvolving pacemaker dysfunctions such as familial sinus rhythm diseases,sick sinus syndrome associated with atrial fibrillation, sinustachycardias, bradycardias and ventricular arrhythmias as well asabnormal ion flux, e.g., CNS disorders and other disorders. For example,chromosome localization of the gene encoding hHAC3 can be used toidentify diseases caused by and associated with the hHAC3. Methods ofdetecting hHAC3 polypeptides are also useful for examining the role ofthe hHAC3 monomers in channel diversity and modulation of channelactivity.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Cation channels with the characteristic of “activation uponhyperpolarization” (also referred to as “hyperpolarization-activated”)have a low probability of opening at cellular resting potentials (fromabout −50 mV to about −80 mV) and have an increasing probability ofopening at more hyperpolarized potentials. Thus a cation channel havingthe characteristic of activation upon hyperpolarization will have agreater probability of opening at −100 mV than at −70 mV. Cationchannels having the characteristic of activation upon hyperpolarizationalso open more quickly at more hyperpolarized potentials. Thus a cationchannel having the characteristic of activation upon hyperpolarizationwill also open more quickly at −100 mV than at −70 mV. A discussion ofactivation by hyperpolarization can be found in Luthi & McCormick,Neuron 21(1):9-12 (1998).

“Cation channels” are a diverse group of proteins that regulate the flowof cations across cellular membranes. The ability of a specific cationchannel to transport particular cations typically varies with thevalency of the cations, as well as the specificity of the given channelfor a particular cation.

“Homomeric channel” refers to a cation channel composed of identicalalpha subunits, whereas “heteromeric channel” refers to a cation channelcomposed of two or more different types of alpha subunits. Bothhomomeric and heteromeric channels can include auxiliary beta subunits.

A “beta subunit” is a polypeptide monomer that is an auxiliary subunitof a cation channel composed of alpha subunits; however, beta subunitsalone cannot form a channel (see, e.g., U.S. Pat. No. 5,776,734). Betasubunits are known, for example, to increase the number of channels byhelping the alpha subunits reach the cell surface, change activationkinetics, and change the sensitivity of natural ligands binding to thechannels. Beta subunits can be outside of the pore region and associatedwith alpha subunits comprising the pore region. They can also contributeto the external mouth of the pore region.

The term “transmembrane domain” refers to the region of the cationchannel subunit polypeptide that spans across the lipid bilayer membraneof the cells. Various families of the cation channels have differentnumbers of transmembrane domains that travel across the cellularmembrane. Structurally, a transmembrane domain starts from the firstamino acid residue of the subunit sequence that enters into the cellularmembrane and ends with the last amino acid residue in the subunitsequence that leaves the cellular membrane.

The phrase “voltage-gated” activity or “voltage-gating” refers to acharacteristic of a HAC channel composed of individual polypeptidemonomers or subunits. Generally, the probability of a voltage-gated HACchannel opening increases as a cell is hyperpolarized. The reversalpotential for HAC channels is primarily determined by the reversalpotentials of the two major permeant cations, sodium and potassium.E_(K), or the reversal potential for potassium, depends on the relativeconcentrations of potassium found inside and outside the cell membrane,and is typically between −60 and −100 mV for mammalian cells. Forexample, E_(K) is the membrane potential at which there is no net flowof potassium ion because the electrical potential (i.e., membranepotential) driving potassium influx is balanced by the concentrationgradient directing potassium efflux. This value is also known as the“reversal potential” or the “Nernst” potential for potassium. Similarly,E_(Na), or the reversal potential for sodium, depends on the relativeconcentration of sodium found inside and outside the cell and istypically near 50 mV. Because HAC channels pass both sodium andpotassium, their reversal potential lies between E_(K) and E_(Na), andis typically −20 to −40 mV. Hyperpolarization activated cation channelsprimarily allow influx of cations because they have greaterprobabilities of being open at membrane potentials more negative thanthis equilibrium potential.

Certain hyperpolarization activated channels such as HAC channels aretypically composed of four subunits and the channel can be heteromericor homomeric. The characteristic of voltage gating can be measured by avariety of techniques for measuring changes in current flow and ion fluxthrough a channel, e.g., by changing the [K⁺] of the external solutionand measuring the activation potential of the channel current (see,e.g., U.S. Pat. No. 5,670,335), by measuring current with patch clamptechniques or voltage clamp under different conditions, and by measuringion flux with radio-labeled tracers or voltage-sensitive dyes underdifferent conditions.

“hHAC3” refers to a polypeptide that is an alpha subunit or monomer of ahyperpolarization-activated cation channel, a member of the HACsubfamily, and a member of the voltage-gated cation channel superfamily. The term hHAC3 therefore refers to conservatively modifiedvariants, polymorphic variants, alleles, mutants that: (1) form cationchannels that are voltage-gated and activated upon hyperpolarization;(2) specifically bind to polyclonal antibodies raised against animmunogen comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 and conservatively modified variants orportions thereof, including the N-terminal region (amino acids 1-50 ofHAC3); (3) have at least about 75% identity to the N-terminal region ofhHAC3 (amino acids 1-50 of HAC3); or (4) are encoded by nucleic acidsthat are amplified by primers that specifically hybridize understringent hybridization conditions to the same sequence as a primer setconsisting of SEQ ID NO:3 and SEQ ID NO:4 or SEQ ID NO:5 and SEQ IDNO:6. Alternatively, hHAC3 can be identified as a cation channel subunitpolypeptide having 90% or more identity to the region defined by aminoacids 640-775 of SEQ ID NO:1.

The phrase “functional effects” in the context of assays for testingcompounds affecting a channel comprising hHAC3 includes thedetermination of any parameter that is indirectly or directly under theinfluence of the channel. It includes changes in ion flux and membranepotential, and also includes other physiologic effects such increases ordecreases of transcription or hormone release.

“Determining the functional effect” refers to examining the effect of acompound that increases or decreases ion flux on a cell or cell membranein terms of cell and cell membrane function. The ion flux can be any ionthat passes through a channel and analogues thereof, e.g., potassium,rubidium, sodium. Preferably, the term refers to the functional effectof the compound on the channels comprising hHAC3, e.g., changes in ionflux including radioisotopes, changes in ion concentration (e.g., Ca²⁺,K⁺, Na⁺) current amplitude, membrane potential, current flow,transcription, protein binding, phosphorylation, dephosphorylation,second messenger concentrations (cAMP, cGMP, Ca²⁺, IP₃), ligand binding,and other physiological effects such as hormone and neurotransmitterrelease, as well as changes in voltage and current. Such functionaleffects can be measured by any means known to those skilled in the art,e.g., patch clamping, voltage-sensitive dyes, whole cell currents,radioisotope efflux, inducible markers, and the like.

“Inhibitors,” “activators” or “modulators” ofhyperpolarization-activated voltage-gated cation channels comprisinghHAC3 refer to inhibitory or activating molecules identified using invitro and in vivo assays for hHAC3 cation channel function. Inhibitorsare compounds that decrease, block, prevent, delay activation,inactivate, desensitize, or down regulate the channel. Activators arecompounds that increase, open, activate, facilitate, enhance activation,sensitize or up regulate channel activity. Such assays for inhibitorsand activators include e.g., expressing hHAC3 in cells or cell membranesand then measuring flux of ions through the channel and determiningchanges in polarization (i.e., electrical potential). To examine theextent of inhibition, samples or assays comprising ahyperpolarization-activated cation channel (e.g., hHAC3) are treatedwith a potential activator or inhibitor and are compared to controlsamples without the inhibitor. Control samples (untreated withinhibitors) are assigned a relative hHAC3 activity value of 100%.Inhibition of channels comprising hHAC3 is achieved when the hHAC3activity value relative to the control is about 90%, preferably 50%,more preferably 25-0%. Activation of channels comprising hHAC3 isachieved when the hHAC3 activity value relative to the control is 110%,more preferably 150%, most preferably at least 200-500% higher or 1000%or higher.

“Biologically active” hHAC3 refers to hHAC3 that comprises a cationchannel having the characteristic of activation upon hyperpolarizationtested as described above.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated hHAC3 nucleic acid is separated from openreading frames that flank the hHAC3 gene and encode proteins other thanhHAC3. The term “purified” denotes that a nucleic acid or protein givesrise to essentially one band in an electrophoretic gel. Particularly, itmeans that the nucleic acid or protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants”. Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition. An example of cationchannel splice variants is discussed in Leicher, et al., J Biol. Chem.273(52):35095-35101 (1998).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins (1984)).

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available(e.g., the polypeptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radio-label into the peptide, and used to detectantibodies specifically reactive with the peptide).

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, or designated conserved region such as the N-terminal region, asmeasured using one of the following sequence comparison algorithms withthe default parameters described below or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the compliment of a testsequence. Preferably, the identity exists over a region that is at leastabout 25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length, morepreferably over the length of the reference amino acid sequence ornucleotide sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. For sequence comparison of nucleicacids and proteins to human HAC3 nucleic acids and proteins, the BLASTand BLAST 2.0 algorithms and the default parameters discussed below areused.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For high stringency hybridization, a positivesignal is at least two times background, preferably 10 times backgroundhybridization. Exemplary high stringency hybridization conditionsinclude: 50% formamide, 5×SSC and 1% SDS incubated at 42° C. or 5×SSCand 1% SDS incubated at 65° C., with a wash in 0.2×SSC and 0.1% SDS at65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)—C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

An “anti-hHAC3” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the hHAC3 gene, cDNA, or asubsequence thereof.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to hHAC3, encoded in SEQ ID NO:1, splice variants, or portionsthereof, can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with hHAC3 and not with other proteins,except for polymorphic variants and alleles of hHAC3. This selection maybe achieved by subtracting out antibodies that cross-react withmolecules such as mouse HAC3 and other HAC3 orthologs. Other humanmembers of the HAC family, such as human HAC1 and 2 can also be used toselect for antibodies that recognize only human HAC3. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast (e.g., Pichia), insect, amphibian, or mammalian cells such asCHO, HeLa and the like, e.g., cultured cells, explants, and cells invivo.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains hHAC3 or nucleic acid encoding hHAC3 protein. Suchsamples include, but are not limited to, tissue isolated from humans.Biological samples may also include sections of tissues such as frozensections taken for histological purposes. A biological sample istypically obtained from a eukaryotic organism, preferably eukaryotessuch as fungi, plants, insects, protozoa, birds, fish, reptiles, andpreferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit,and most preferably a primate such as chimpanzees or humans.

III. Isolating the Gene Encoding hHAC3

-   A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

-   B. Cloning Methods for the Isolation of Nucleotide Sequences    Encoding hHAC3

In general, the nucleic acid sequences encoding hHAC3 and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. For example, hHAC3 sequences are typicallyisolated from human nucleic acid (genomic or cDNA) libraries byhybridizing with a nucleic acid probe or polynucleotide, the sequence ofwhich can be derived from SEQ ID NO:2.

A suitable tissue from which hHAC3 RNA and cDNA can be isolated is theputamen, thalamus, caudate nucleus, medulla, occipital lobe, substantianigra, spinal cord, and fetal brain. See Example 1 for a complete listof the tissues in which hHAC3 is expressed.

Amplification techniques using primers can also be used to amplify andisolate hHAC3 from DNA or RNA. The following primers can also be used toamplify a sequence of hHAC3: CAGCCATGGAGGCAGAGCAGCGGC, (SEQ ID NO:3)GGAGGAGATCTTTCACATGACATACGAC, (SEQ ID NO:4) AGTAGGATCCATCGGTGAGGCGTG,(SEQ ID NO:5) TTACATGTTGGCAGAAAGCTGGAGACC. (SEQ ID NO:6)

These primers can be used, e.g., to amplify either the full lengthsequence or a probe of one to several hundred nucleotides, which is thenused to screen a human library for full-length hHAC3.

Nucleic acids encoding hHAC3 can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:1.

Human HAC3 polymorphic variants and alleles that are substantiallyidentical SEQ ID NO:2 can be isolated using hHAC3 nucleic acid probesand oligonucleotides under stringent hybridization conditions, byscreening libraries. Alternatively, expression libraries can be used toclone hHAC3 and hHAC3 polymorphic variants and alleles by detectingexpressed homologs immunologically with antisera or purified antibodiesmade against hHAC3 or portions thereof (e.g., the N-terminal region,amino acids 1-50 of HAC3), which also recognize and selectively bind tothe hHAC3 homolog.

To make a cDNA library, one should choose a source that is rich in hHAC3mRNA, e.g., tissue such as the thalamus, medulla or fetal brain. ThemRNA is then made into cDNA using reverse transcriptase, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating hHAC3 nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of hHAC3directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.Degenerate oligonucleotides can be designed to amplify hHAC3 homologsusing the sequences provided herein. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of hHAC3encoding mRNA in physiological samples, for nucleic acid sequencing, orfor other purposes. Genes amplified by the PCR reaction can be purifiedfrom agarose gels and cloned into an appropriate vector.

Gene expression of hHAC3 can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology and the like.

Synthetic oligonucleotides can be used to construct recombinant hHAC3genes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the hHAC3 gene. The specificsubsequence is then ligated into an expression vector.

The gene for hHAC3 is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

-   C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding hHAC3, one typically subclones hHAC3 into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systemsfor expressing the hHAC3 protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the hHAC3 encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding hHAC3and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the CMV promoter, SV40early promoter, SV40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal. Inducible expression vectors are oftenchosen if expression of the protein of interest is detrimental toeukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with ahHAC3 encoding sequence under the direction of the polyhedrin promoteror other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of hHAC3protein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressinghHAC3.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofhHAC3, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of hHAC3 Polypeptides

Either naturally occurring or recombinant hHAC3 can be purified for usein functional assays. Naturally occurring hHAC3 monomers can bepurified, e.g., from mouse or human tissue such as thalamus, medulla orfetal brain tissue and any other source of a hHAC3 homolog. RecombinanthHAC3 monomers can be purified from any suitable expression system.

The hHAC3 monomers may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant hHAC3 monomersare being purified. For example, proteins having established molecularadhesion properties can be reversible fused to the hHAC3 monomers. Withthe appropriate ligand, the hHAC3 monomers can be selectively adsorbedto a purification column and then freed from the column in a relativelypure form. The fused protein is then removed by enzymatic activity.Finally the hHAC3 monomers could be purified using immunoaffinitycolumns.

-   A. Purification of hHAC3 Monomers from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of the hHAC3monomers inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human HAC3 monomers areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify the hHAC3 monomers from bacteriaperiplasm. After lysis of the bacteria, when the hHAC3 monomers areexported into the periplasm of the bacteria, the periplasmic fraction ofthe bacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

-   B. Standard Protein Separation Techniques for Purifying the hHAC3    Monomers

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the hHAC3 monomers can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

The hHAC3 monomers can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

V. Immunological Detection of hHAC3

In addition to the detection of hHAC3 genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect the hHAC3 monomers. Immunoassays can be used to qualitatively orquantitatively analyze the hHAC3 monomers. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

-   A. Antibodies to hHAC3 Monomers

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the hHAC3 monomers are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of immunogens comprising portions of hHAC3 monomers may be usedto produce antibodies specifically reactive with hHAC3 monomers. Forexample, recombinant hHAC3 monomers or an antigenic fragment thereofsuch as amino acids 1-50 or amino acids 640-775 of SEQ ID NO:1, can beisolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-hHAC3proteins or even other related proteins from other organisms (e.g.,other HAC family members), using a competitive binding immunoassay.Specific polyclonal antisera and monoclonal antibodies will usually bindwith a K_(D) of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most preferably, 0.01 μMor better.

Once the specific antibodies against a hHAC3 are available, the hHAC3can be detected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

-   B. Immunological Binding Assays

The hHAC3 can be detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7^(th) ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the hHAC3 or an antigenicsubsequence thereof). The antibody (e.g., anti-hHAC3) may be produced byany of a number of means well known to those of skill in the art and asdescribed above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled hHAC3 polypeptide ora labeled anti-hHAC3 antibody. Alternatively, the labeling agent may bea third moiety, such a secondary antibody, which specifically binds tothe antibody/hHAC3 complex (a secondary antibody is typically specificto antibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-competitive Assay Formats

Immunoassays for detecting the hHAC3 in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-hHAC3 subunit antibodies can bebound directly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture hHAC3 present in the test sample.The hHAC3 monomers are thus immobilized and then bound by a labelingagent, such as a second hHAC3 antibody bearing a label. Alternatively,the second antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

Competitive Assay Formats

In competitive assays, the amount of the hHAC3 present in the sample ismeasured indirectly by measuring the amount of known, added (exogenous)hHAC3 displaced (competed away) from an anti-hHAC3 antibody by theunknown hHAC3 present in a sample. In one competitive assay, a knownamount of the hHAC3 is added to a sample and the sample is thencontacted with an antibody that specifically binds to the hHAC3. Theamount of exogenous hHAC3 bound to the antibody is inverselyproportional to the concentration of the hHAC3 present in the sample. Ina particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of hHAC3 bound to the antibody may bedetermined either by measuring the amount of hHAC3 present in ahHAC3/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of hHAC3 may be detected byproviding a labeled hHAC3 molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known hHAC3 is immobilized on a solid substrate. A knownamount of anti-hHAC3 antibody is added to the sample, and the sample isthen contacted with the immobilized hHAC3. The amount of anti-hHAC3antibody bound to the known immobilized hHAC3 is inversely proportionalto the amount of hHAC3 present in the sample. Again, the amount ofimmobilized antibody may be detected by detecting either the immobilizedfraction of antibody or the fraction of the antibody that remains insolution. Detection may be direct where the antibody is labeled orindirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations for hHAC3. For example, a protein atleast partially encoded by SEQ ID NO:2 or an immunogenic region thereof,such as the N-terminal region (amino acids 1-50), can be immobilized toa solid support. Other proteins such as other HAC family members, e.g.,mouse HAC3 or human HAC1 or HAC2, are added to the assay so as tocompete for binding of the antisera to the immobilized antigen. Theability of the added proteins to compete for binding of the antisera tothe immobilized protein is compared to the ability of the hHAC3 encodedby SEQ ID NO:1 to compete with itself. The percent crossreactivity forthe above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the addedproteins listed above are selected and pooled. The cross-reactingantibodies are optionally removed from the pooled antisera byimmunoabsorption with the added considered proteins, e.g., distantlyrelated homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of hHAC3, to theimmunogen protein. In order to make this comparison, the two proteinsare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding of the antisera tothe immobilized protein is determined. If the amount of the secondprotein required to inhibit 50% of binding is less than 10 times theamount of the protein encoded by hHAC3 that is required to inhibit 50%of binding, then the second protein is said to specifically bind to thepolyclonal antibodies generated to the respective hHAC3 immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of the hHAC3 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind hHAC3. The anti-hHAC3 antibodies specificallybind to hHAC3 on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-hHAC3 antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize hHAC3, orsecondary antibodies that recognize anti-hHAC3 antibodies.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of hHAC3

-   A. Assays

Human HAC3 monomers and hHAC3 alleles and polymorphic variants aresubunits of hyperpolarization-activated cation channels. The activity ofa cation channel comprising hHAC3 can be assessed using a variety of invitro and in vivo assays, e.g., measuring current, measuring membranepotential, measuring ion flux, e.g., potassium, sodium, guanidinium, andrubidium, measuring potassium or other cation concentration, measuringsecond messengers and transcription levels, using potassium-dependentyeast growth assays, measuring ligand binding, and using e.g.,voltage-sensitive dyes, radioactive tracers, and patch-clampelectrophysiology. hHAC polypeptides and channels can be attached to asolid substrate, in solution, or expressed in a cell or cell membranethat is attached to a solid substrate or in solution. Channels made ofHAC family members are typically blocked by about 2 mM cesium.

Furthermore, such assays can be used to test for inhibitors andactivators of channels comprising hHAC3. Such modulators of ahyperpolarization-activated cation channel are useful for treatingvarious disorders involving cation channels. Treatment of dysfunctionsinclude pacemaker dysfunctions such as familial sinus rhythm diseases,sick sinus syndrome associated with atrial fibrillation, and ventriculararrhythmias, memory and learning disorders, sleeping disorders, bipolardisease, schizophrenia, as well as CNS disorders such as migraines,hearing and vision problems, seizures, and as neuroprotective agents(e.g., to prevent stroke). Such modulators are also useful forinvestigation of the channel diversity provided by hHAC3 and theregulation/modulation of cation channel activity provided by hHAC3.

Modulators of the hyperpolarization-activated cation channels are testedusing biologically active hHAC3, either recombinant or naturallyoccurring. Human HAC3 can be isolated, expressed in a cell, or expressedin a membrane derived from a cell. In such assays, hHAC3 is expressedalone to form a homomeric cation channel or is co-expressed with asecond alpha subunit (e.g., HAC1 or HAC2) so as to form a heteromericcation channel. HAC can also be expressed with additional beta subunits.Modulation is tested using one of the in vitro or in vivo assaysdescribed above. Samples or assays that are treated with a potentialcation channel inhibitor or activator are compared to control sampleswithout the test compound, to examine the extent of modulation. Controlsamples (untreated with activators or inhibitors) are assigned arelative cation channel activity value of 100. Inhibition of channelscomprising hHAC3 is achieved when the cation channel activity valuerelative to the control is about 90%, preferably 50%, more preferably25-0%. Activation of channels comprising hHAC3 is achieved when thecation channel activity value relative to the control is 110%, morepreferably 150%, more preferable 200% higher. Compounds that increasethe flux of ions will cause a detectable increase in the ion currentdensity by increasing the probability of a channel comprising hHAC3being open, by decreasing the probability of it being closed, byincreasing conductance through the channel, and/or by allowing thepassage of ions.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the cation channel comprising hHAC3. A preferred means todetermine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques, e.g., the “cell-attached” mode, the“inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman etal., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents areconveniently determined using the standard methodology (see, e.g., Hamilet al., PFlugers. Archiv. 391:85 (1981). Other known assays include:radiolabeled rubidium, sodium, or guanidinium flux assays andfluorescence assays using voltage-sensitive dyes or ion-sensitive dyes(see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75(1988); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Holevinskyet al., J. Membrane Biology 137:59-70 (1994)). Assays for compoundscapable of inhibiting or increasing potassium flux through the channelproteins comprising hHAC3 can be performed by application of thecompounds to a bath solution in contact with and comprising cells havinga channel of the present invention (see, e.g., Blatz et al., Nature323:718-720 (1986); Park, J. Physiol. 481:555-570 (1994)). Generally,the compounds to be tested are present in the range from 1 pM to 100 mM.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of cations such as potassium, sodium, guanidinium, or rubidiumions. The cations can be measured in a variety of standard ways. Theycan be measured directly by concentration changes of the ions orindirectly by membrane potential or by radio-labeling of the ions.Ligand binding to the channel or polypeptide can be measured by standardassays known to those of skill in the art. Consequences of the testcompound on ion flux can be quite varied. Accordingly, any suitablephysical, chemical or physiological change can be used to assess theinfluence of a test compound on the channels of this invention. Theeffects of a test compound can be measured by a toxin binding assay.When the functional consequences are determined using intact cells oranimals, one can also measure a variety of effects such as transmitterrelease (e.g., dopamine), hormone release (e.g., insulin),transcriptional changes to both known and uncharacterized geneticmarkers (e.g., northern blots), cell volume changes (e.g., in red bloodcells), immunoresponses (e.g., T cell activation), changes in cellmetabolism such as cell growth or pH changes, and changes inintracellular second messengers such as Ca2⁺, or cyclic nucleotides.

Preferably, the HAC3 that is a part of the hyperpolarization-activatedcation channel used in the assay will have the sequence displayed in SEQID NO:1 or a conservatively modified variant thereof. Alternatively, theHAC3 of the assay will be derived from a eukaryote and include an aminoacid subsequence having amino acid sequence identity to hHAC3.Generally, the amino acid sequence identity will be at least 96%,preferably at least 98%, most preferably at least 99%.

Human HAC3 orthologs will generally confer substantially similarproperties on a channel comprising such hHAC3, as described above. In apreferred embodiment, the cell placed in contact with a compound that issuspected to be a hHAC3 homolog is assayed for increasing or decreasingion flux in a eukaryotic cell, e.g., an oocyte of Xenopus (e.g., Xenopuslaevis) or a mammalian cell such as a CHO or HeLa cell. Channels thatare affected by compounds in ways similar to hHAC3 are consideredhomologs or orthologs of hHAC3.

-   B. Modulators

The compounds tested as modulators of HAC channels comprising a humanHAC3 subunit can be any small chemical compound, or a biological entity,such as a protein, sugar, nucleic acid or lipid. Alternatively,modulators can be genetically altered versions of a human HAC3 subunit.Typically, test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan.18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressinga HAC3 channel comprising a human HAC3 subunit is attached to a solidphase substrate. In the high throughput assays of the invention, it ispossible to screen up to several thousand different modulators orligands in a single day. In particular, each well of a microtiter platecan be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100- about 1500 different compounds. It is possible to assayseveral different plates per day; assay screens for up to about6,000-20,000 different compounds is possible using the integratedsystems of the invention. More recently, microfluidic approaches toreagent manipulation have been developed.

-   C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provide soluble assays using potassiumchannels comprising hHAC3; a membrane comprising a channel comprisinghHAC3, or a cell or tissue expressing channels comprising hHAC3, eithernaturally occurring or recombinant. In another embodiment, the inventionprovides solid phase based in vitro assays in a high throughput format,where hHAC3 channel attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100- about 1500different compounds. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds is possible using the integrated systems of theinvention.

The channel of interest, or a cell or membrane comprising the channel ofinterest can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest (e.g., the taste transduction molecule of interest)is attached to the solid support by interaction of the tag and the tagbinder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

VII. Computer Assisted Drug Design Using hHAC3

Yet another assay for compounds that modulate the activities of hHAC3involves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of hHAC3 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands or other cationchannel subunits. These regions are then used to identify ligands thatbind to the protein or region where hHAC3 interacts with other cationchannel subunits.

The three-dimensional structural model of the protein is generated byentering channel protein amino acid sequences of at least 25-75 aminoacid residues or corresponding nucleic acid sequences encoding a hHAC3monomer into the computer system. The amino acid sequence of each of themonomers is selected from the group consisting of SEQ ID NO:1 and aconservatively modified versions thereof. The amino acid sequencerepresents the primary sequence or subsequence of each of the proteins,which encodes the structural information of the protein. At least 25-75residues of the amino acid sequence (or a nucleotide sequence encoding25-75 amino acids) are entered into the computer system from computerkeyboards, computer readable substrates that include, but are notlimited to, electronic storage media (e.g., magnetic diskettes, tapes,cartridges, and chips), optical media (e.g., CD ROM), informationdistributed by internet sites, and by RAM. The three-dimensionalstructural model of the channel protein is then generated by theinteraction of the amino acid sequence and the computer system, usingsoftware known to those of skill in the art. The resultingthree-dimensional computer model can then be saved on a computerreadable substrate.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the monomer and the heteromeric potassium channel proteincomprising four monomers. The software looks at certain parametersencoded by the primary sequence to generate the structural model. Theseparameters are referred to as “energy terms,” or anisotropic terms andprimarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of hHAC3 protein to identify ligands that bind to hHAC3. Bindingaffinity between the protein and ligands is determined using energyterms to determine which ligands have an enhanced probability of bindingto the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of hHAC3 genes. Suchmutations can be associated with disease states. Once the variants areidentified, diagnostic assays can be used to identify patients havingsuch mutated genes associated with disease states. Identification of themutated hHAC3 genes involves receiving input of a first nucleic acid,e.g., SEQ ID NO:2, or an amino acid sequence encoding hHAC3, selectedfrom the group consisting of SEQ ID NO:1, and a conservatively modifiedversions thereof. The sequence is entered into the computer system asdescribed above. The first nucleic acid or amino acid sequence is thencompared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in hHAC3 genes, and mutations associatedwith disease states. The first and second sequences described above canbe saved on a computer readable substrate.

Human HAC3 monomers and the hyperpolarization-activated cation channelscontaining these hHAC3 monomers can be used with high densityoligonucleotide array technology (e.g., GeneChip™) to identify homologsand polymorphic variants of hHAC3 in this invention. In the case wherethe homologs being identified are linked to a known disease, they can beused with GeneChip™ as a diagnostic tool in detecting the disease in abiological sample, see, e.g., Gunthand et al., AIDS Res. Hum.Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart etal., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

VIII. Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of hHAC3 for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid for hHAC3, under thecontrol of a promoter, then expresses a hHAC3 monomer of the presentinvention, thereby mitigating the effects of absent, partialinactivation, or abnormal expression of the hHAC3 gene. The compositionsare administered to a patient in an amount sufficient to elicit atherapeutic response in the patient. An amount adequate to accomplishthis is defined as “therapeutically effective dose or amount.”

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and viral infection in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy 1: 13-26 (1994)).

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A large number ofdelivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™. Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801(1994); Muzyczka, J.Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci.U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeuticvector used in a gene therapy trial. (Blaese et al., Science 270:475-480(1995)). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors. (Ellem et al., Immunol Immunother.44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 241:5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., Proc. Natl. Acad. Sci. U.S.A.92:9747-9751 (1995), reported that Moloney murine leukemia virus can bemodified to express human heregulin fused to gp70, and the recombinantvirus infects certain human breast cancer cells expressing humanepidermal growth factor receptor. This principle can be extended toother pairs of virus expressing a ligand fusion protein and target cellexpressing a receptor. For example, filamentous phage can be engineeredto display antibody fragments (e.g., FAB or Fv) having specific bindingaffinity for virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA), and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The nucleicacids are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch nucleic acids are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

IX. Pharmaceutical Compositions

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid, protein,modulatory compounds or transduced cell), as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989). Administration can be in any convenient manner,e.g., by injection, oral administration, inhalation, transdermalapplication, or rectal administration.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, usuallysucrose and acacia or tragacanth, as well as pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the active ingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of the HAC3 channels comprising a human HAC3 alphasubunit, the physician evaluates circulating plasma levels of thevector, vector toxicities, progression of the disease, and theproduction of anti-vector antibodies. In general, the dose equivalent ofa naked nucleic acid from a vector is from about 1 μg to 100 μg for atypical 70 kilogram patient, and doses of vectors which include aretroviral particle are calculated to yield an equivalent amount oftherapeutic nucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

Transduced cells are prepared for reinfusion according to establishedmethods (see Abrahamsen et al., J Clin. Apheresis 6:48-53 (1991); Carteret al., J. Clin. Apheresis 4:113-117 (1998); Aebersold et al., J.Immunol. Meth. 112:1-7 (1998); Muul et al., J. Immunol. Methods101:171-181 (1987); and Carter et al., Transfusion 27:362-365 (1987)).After a period of about 2-4 weeks in culture, the cells should numberbetween 1×10⁸ and 1×10¹². In this regard, the growth characteristics ofcells vary from patient to patient and from cell type to cell type.About 72 hours prior to reinfusion of the transduced cells, an aliquotis taken for analysis of phenotype, and percentage of cells expressingthe therapeutic agent.

X. Kits

Human HAC3 and its homologs are useful tools for examining expressionand regulation of hyperpolarization-activated cation channels. HumanHAC3-specific reagents that specifically hybridize to hHAC3 nucleicacid, such as hHAC3 probes and primers, and hHAC3-specific reagents thatspecifically bind to the hHAC3 protein, e.g., hHAC3 antibodies are usedto examine expression and regulation.

Nucleic acid assays for the presence of hHAC3 DNA and RNA in a sampleinclude numerous techniques are known to those skilled in the art, suchas Southern analysis, northern analysis, dot blots, RNase protection, S1analysis, amplification techniques such as PCR and LCR, and in situhybridization. In in situ hybridization, for example, the target nucleicacid is liberated from its cellular surroundings in such as to beavailable for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the art of in situhybridization: Singer et al., Biotechniques 4:230-250 (1986); Haase etal., Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic AcidHybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, hHAC3 protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant hHAC3monomers) and a negative control.

The present invention also provides for kits for screening modulators ofthe heteromeric potassium channels. Such kits can be prepared fromreadily available materials and reagents. For example, such kits cancomprise any one or more of the following materials: hHAC3 monomers,reaction tubes, and instructions for testing the activities ofhyperpolarization-activated cation channels containing hHAC3. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user. For example, the kit can be tailored forin vitro or in vivo assays for measuring the activity of ahyperpolarization-activated cation channel comprising a hHAC3 monomer.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLE

The following example is provided by way of illustration only and not byway of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Isolation of Nucleic Acids Encoding hHAC3 and FunctionalAnalysis of Hyperpolarization-activated Cation Channels Containing hHAC3

Using PCR and primers, according to standard conditions, hHAC3 wasamplified from a human hippocampus cDNA library. The followingdegenerate primers were used for amplification of hHAC3: (1)5′-TGGGAGGAGATCTTYCAYATGACNTAYGA-3 (SEQ ID NO: 7) (2)5′-CGTCTCGAATGCCCKNCKCATCATNGG-3 (SEQ ID NO: 8)

Primers (1) and (2) were used together to amplify portions of the regionthat encode the S4 and putative cyclic-nucleotide binding domains ofhyperpolarization-activated cation channels. PCR conditions were asfollows: 95 degrees for 15 seconds, 60-40 degrees for 15 seconds, 72degrees for 45 seconds. The reaction was run for 40 cycles.

5′ and 3′ RACE PCR was subsequently used to clone the complete ends ofthe hHAC3 gene from hippocampal cDNA. The Clontech Marathon RACE kit wasused for this procedure. A gene specific oligo is used in combinationwith a non-selective oligo tagged to the cDNA end. Two rounds of 5′ RACEwere performed. In the first round, the gene specific primer wasCCTGCTGCCCATAGCCAATGCACAGC (SEQ ID NO:9). In the second round, the firstreaction was reamplified with the nested gene-specific primerGCACCACGAACTGCAGACAGCCATC (SEQ ID NO:10). For the 3′ RACE, four nestedrounds were performed with the following gene specific primers:GTTCTCACCAAGCTGCGCTTTGAGGTC (SEQ ID NO:11) CCAGCATGGGCTGCTCAGTGTGCTG(SEQ ID NO:12) GCCCACTCTCAGCCTCCCAACCCTC (SEQ ID NO:13)CCCAACCAAGCTTGCCTCAGCGGGCAACAGGCGAT (SEQ ID NO:14) GG

The sequence of the degernerate PCR product and the 5′ and 3′ RACEproduct were overlapped to produce a contiguous HAC3 sequence spanningthe entire coding region. The entire coding region can be amplified in asingle fragment using primers SEQ ID NO:3 and 6. The nucleotide andamino acid sequences of hHAC3 are provided, respectively, in SEQ ID NO:2 and SEQ ID NO: 1.

Human HAC3 monomer was expressed according to standard methodology inoocytes to demonstrate its ability to form cation channels. HAC3expresses a cation channel that opens upon hyperpolarization whenexpressed in Xenopus oocytes. The current activates over several secondsat voltage steps more hyperpolarized than −80 mV, with little or norinactivation. The reversal potential of the current lies between −30 and−40 mV, indicating that HAC3 is a classic I_(h) channel that passes bothsodium and potassium. HAC3 is distinct from other I_(h) channels in thatits activation is particularly slow and occurs at more hyperpolarizedpotentials.

Human HAC3 expression patterns were analyzed using northern blots andmRNA dot blots. Human HAC3 expression was especially high in theputamen, thalamus, caudate nucleus, medulla, occipital lobe, substantianigra, spinal cord and fetal brain.

Human HAC3 was also expressed at moderate levels in several tissues,such as the amygdala, cerebellum, cerebral cortex, frontal lobe,hippocampus, temporal lobe, nucleus accumbens, heart, stomach, pancreas,pituitary gland, liver and appendix. The colon and small intestinedisplayed much higher expression when measured with mRNA dot blots thanwith northern blots. Low to trace levels of expression were found intissues such as prostate, testis, adrenal, thyroid gland, salivarygland, kidney, spleen, thymus, bone marrow, lung trachea, placenta,aorta, skeletal muscles, bladder, uterus, ovary, mammary glands,peripheral leukocytes and many fetal tissues. Sequence Listing SEQ IDNO: 1--human HAC amino acid sequenceMEAEQRPAAGASEGATPGLEAVPPVAPPPATAASGPIPKSGPEPKRRHLGTLLQPTVNKFSLRVFGSHKAVEIEQERVKSAGAWIIHPYSDFRFYWDLIMLLLMVGNLIVLPVGITFFKEENSPPWIVFNVLSDTFFLLDLVLNFRTGIVVEEGAEILLAPRAIRTRYLRTWFLVDLISSIPVDYIFLVVELEPRLDAEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVVRIFNLIGMMLLLCHWDGCLQFLVPMLQDFPPDCWVSINHMVNHSWGRQYSHALFKAMSHMLCIGYGQQAPVGMPDVWLTMLSMIVGATCYAMFIGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPADTRQRIHEYYEHRYQGKMFDEESILGELSEPLREEIINFTCRGLVAHMPLFAHADPSFVTAVLTKLRFEVFQPGDLVVREGSVGRKMYFIQHGLLSVLARGARDTRLTDGSYFGEICLLTRGRRTASVRADTYCRLYSLSVDHFNAVLEEFPMMRRAFETVAMDRLLRIGKKNSILQRKRSEPSPGSSGGIMEQHLVQHDRDMARGVRGRAPSTGAQLSGKPVLWEPLVHAPLQAAAVTSNVAIALTHQRGPLPLSPDSPATLLARSAWRSAGSPASPLVPVRAGPWASTSRLPAPPARTLHASLSRAGRSQVSLLGPPPGGGGRRLGPRGRPLSASQPSLPQRATGDGSPGRKGSGSERLPPSGLLAKPPRTAQPPRPPVPEPATPRGLQLSANM SEQ IDNO:2--human HAC3 nucleotide sequenceATGGAGGCAGAGCAGCGGCCGGCGGCGGGGGCCAGCGAAGGGGCGACCCCTGGACTGGAGGCGGTGCCTCCCGTTGCTCCCCCGCCTGCGACCGCGGCCTCAGGTCCGATCCCCAAATCTGGGCCTGAGCCTAAGAGGAGGCACCTTGGGACGCTGCTCCAGCCTACGGTCAACAAGTTCTCCCTTCGGGTGTTCGGCAGCCACAAAGCAGTGGAAATCGAGCAGGAGCGGGTGAAGTCAGCGGGGGCCTGGATCATCCACCCCTACAGCGACTTCCGGTTTTACTGGGACCTGATCATGCTGCTGCTGATGGTGGGGAACCTCATCGTCCTGCCTGTGGGCATCACCTTCTTCAAGGAGGAGAACTCCCCGCCTTGGATCGTCTTCAACGTATTGTCTGATACTTTCTTCCTACTGGATCTGGTGCTCAACTTCCGAACGGGCATCGTGGTGGAGGAGGGTGCTGAGATCCTGCTGGCACCGCGGGCCATCCGCACGCGCTACCTGCGCACATGGTTCCTGGTTGACCTCATCTCTTCTATCCCTGTGGATTACATCTTCCTAGTGGTGGAGCTGGAGCCACGGTTGGACGCTGAGGTCTACAAAACGGCACGGGCCCTACGCATCGTTCGCTTCACCAAGATCCTAAGCCTGCTGAGGCTGCTCCGCCTCTCCCGCCTCATCCGCTACATACACCAGTGGGAGGAGATCTTTCACATGACCTATGACCTGGCCAGTGCTGTGGTTCGCATCTTCAACCTCATTGGGATGATGCTGCTGCTATGTCACTGGGATGGCTGTCTGCAGTTCCTGGTGCCCATGCTGCAGGACTTCCCTCCCGACTGCTGGGTCTCCATCAACCACATGGTGAACCACTCGTGGGGCCGCCAGTATTCCCATGCCCTGTTCAAGGCCATGAGCCACATGCTGTGCATTGGCTATGGGCAGCAGGCACCTGTAGGCATGCCCGACGTCTGGCTCACCATGCTCAGCATGATCGTAGGTGCCACATGCTACGCCATGTTCATCGGCCATGCCACGGCACTCATCCAGTCCCTGGACTCTTCCCGGCGTCAGTACCAGGAGAAGTACAAGCAGGTGGAGCAGTACATGTCCTTCCACAAGCTGCCAGCAGACACGCGGCAGCGCATCCACGAGTACTATGAGCACCGCTACCAGGGCAAGATGTTCGATGAGGAAAGCATCCTGGGCGAGCTGAGCGAGCCGCTTCGCGAGGAGATCATTAACTTCACCTGTCGGGGCCTGGTGGCCCACATGCCGCTGTTTGCCCATGCCGACCCCAGCTTCGTCACTGCAGTTCTCACCAAGCTGCGCTTTGAGGTCTTCCAGCCGGGGGATCTCGTGGTGCGTGAGGGCTCCGTGGGGAGGAAGATGTACTTCATCCAGCATGGGCTGCTCAGTGTGCTGGCCCGCGGCGCCCGGGACACACGCCTCACCGATGGATCCTACTTTGGGGAGATCTGCCTGCTAACTAGGGGCCGGCGCACAGCCAGTGTTCGGGCTGACACCTACTGCCGCCTTTACTCACTCAGCGTGGACCATTTCAATGCTGTGCTTGAGGAGTTCCCCATGATGCGCCGGGCCTTTGAGACTGTGGCCATGGATCGGCTGCTCCGCATCGGCAAGAAGAATTCCATACTGCAGCGGAAGCGCTCCGAGCCAAGTCCAGGCAGCAGTGGTGGCATCATGGAGCAGCACTTGGTGCAACATGACAGAGACATGGCTCGGGGTGTTCGGGGTCGGGCCCCGAGCACAGGAGCTCAGCTTAGTGGAAAGCCAGTACTGTGGGAGCCACTGGTACATGCGCCCCTTCAGGCAGCTGCTGTGACCTCCAATGTGGCCATTGCCCTGACTCATCAGCGGGGCCCTCTGCCCCTCTCCCCTGACTCTCCAGCCACCCTCCTTGCTCGCTCTGCTTGGCGCTCAGCAGGCTCTCCAGCTTCCCCGCTGGTGCCCGTCCGAGCTGGCCCATGGGCATCCACCTCCCGCCTGCCCGCCCCACCTGCCCGAACCCTGCACGCCAGCCTATCCCGGGCAGGGCGCTCCCAGGTCTCCCTGCTGGGTCCCCCTCCAGGAGGAGGTGGACGGCGGCTAGGACCTCGGGGCCGCCCACTCTCAGCCTCCCAACCCTCTCTGCCTCAGCGGGCAACAGGCGATGGCTCTCCTGGGCGTAAGGGATCAGGAAGTGAGCGGCTGCCTCCCTCAGGGCTCCTGGCCAAACCTCCAAGGACAGCCCAGCCCCCCAGGCCACCAGTGCCTGAGCCAGCCACACCCCGGGGTCTCCAGCTTTCTGCCAACATGTAA

1-19. (canceled)
 20. An antibody that selectively binds to an isolatedpolypeptide comprising an alpha subunit of a cation channel, thepolypeptide: (i) forming, with at least one additional HAC alphasubunit, a cation channel having the characteristic of activation uponhyperpolarization; and (ii) having an amino acid sequence that hasgreater than about 96% identity to SEQ ID NO:1.
 21. The antibody ofclaim 20, wherein the polypeptide has an amino acid sequence of SEQ IDNO:1. 22-39. (canceled)
 40. The antibody of claim 20, wherein thepolypeptide comprises an alpha subunit of a homomeric cation channel.41. The antibody of claim 20, wherein the polypeptide comprises an alphasubunit of a heteromeric cation channel.
 42. The antibody of claim 20,wherein the antibody is a monoclonal antibody.